152163 Nucleic Acids Research, 2016, Vol. 44, No.

1
doi: 10.1093/nar/gkv900

Published online 17 September 2015

Guanine quadruplex structures localize to

heterochromatin
Roland F. Hoffmann1 , Yuri M. Moshkin2 , Stijn Mouton1 , Nicola A. Grzeschik3 , Ruby
D. Kalicharan3 , Jeroen Kuipers3 , Anouk H.G. Wolters3 , Kazuki Nishida4 , Aleksander
V. Romashchenko2,5 , Jan Postberg6 , Hans Lipps7 , Eugene Berezikov1,5 , Ody C.M. Sibon3 ,
Ben N.G. Giepmans3 and Peter M. Lansdorp1,8,*
1

Received June 26, 2015; Revised August 19, 2015; Accepted August 21, 2015

ABSTRACT
Increasing amounts of data support a role for guanine quadruplex (G4) DNA and RNA structures in
various cellular processes. We stained different organisms with monoclonal antibody 1H6 specific for
G4 DNA. Strikingly, immuno-electron microscopy
showed exquisite specificity for heterochromatin.
Polytene chromosomes from Drosophila salivary
glands showed bands that co-localized with heterochromatin proteins HP1 and the SNF2 domaincontaining protein SUUR. Staining was retained in
SUUR knock-out mutants but lost upon overexpression of SUUR. Somatic cells in Macrostomum lignano were strongly labeled, but pluripotent stem
cells labeled weakly. Similarly, germline stem cells
in Drosophila ovaries were weakly labeled compared
to most other cells. The unexpected presence of G4
structures in heterochromatin and the difference in
G4 staining between somatic cells and stem cells
with germline DNA in ciliates, flatworms, flies and
mammals point to a conserved role for G4 structures
in nuclear organization and cellular differentiation.
INTRODUCTION
The mechanisms and processes that govern the organization
of DNA in eukaryotic chromosomes and cells are incom* To

pletely understood (1). In nuclei dense areas of heterochromatin can typically be distinguished next to more open areas of euchromatin (2,3). How these different morphologies
relate to known chromatin features such as histone modifications, DNA methylation and non-coding RNA is subject of intense research efforts (4,5). Similarly, the organization of chromatin in mitotic chromosomes remains unclear.
Recent data challenge the classic model in which 10-nm
DNA fibers are folded into 30-nm chromatin fibers (6,7). Instead, it was proposed that packaging of 10-nm DNA fibers
is achieved in a fractal manner whereby DNA fibers fold
back and interact at many different levels (8).
In vitro, single stranded guanine-rich RNA or DNA
readily adopts higher order structures known as guanine
quadruplex (G4) structures (9). Extensive studies have documented that the formation and stability of G4 structures
under physiological conditions depends on many factors including the concentrations of various ions in the nucleus
(10,11). G4 formation could furthermore depend on the
presence of specific proteins and their post-translational
modifications, non-coding RNAs and factors that influence the stability of duplex DNA such as cytosine methylation (12) and DNA supercoiling (13). Sequences capable
of forming G4 DNA are abundant in human DNA (14
16) and such sequences are enriched in promoters and the
first intron of many genes and at telomeres (17). Whereas
G4 RNA could presumably readily form in specific G-rich
transcripts, it is typically assumed that guanine-rich DNA
must dissociate from its complementary C-rich sequences

whom correspondence should be addressed. Tel: +31 50 361 7300; Fax: +31 50 361 7300; Email: p.m.lansdorp@umcg.nl


C The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which
permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact
journals.permissions@oup.com

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European Research Institute for the Biology of Ageing, University of Groningen, University Medical Centre
Groningen, A. Deusinglaan 1, NL-9713 AV Groningen, The Netherlands, 2 Department of Biochemistry, Erasmus
University Medical Center, Dr. Molewaterplein 50, NL-3015 GE Rotterdam, The Netherlands, 3 Department of Cell
Biology, University of Groningen, University Medical Centre Groningen, A. Deusinglaan 1, NL-9713 AV Groningen,
The Netherlands, 4 Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan, 5 Institute of Cytology and Genetics,
Siberian Branch of the Russian Academy of Sciences, Novosibirsk 630090, Russia, 6 Helios Medical Centre
Wuppertal, Paediatrics Centre, Witten/Herdecke University, Wuppertal, Germany, 7 Institute of Cell Biology, Centre for
Biomedical Education and Research, Witten/Herdecke University, Witten, Germany and 8 Terry Fox Laboratory,
British Columbia Cancer Agency and Department of Medicine, University of British Columbia Vancouver, BC, V5Z
1L3, Canada

Nucleic Acids Research, 2016, Vol. 44, No. 1 153

MATERIALS AND METHODS

General
Fluorescent secondary antibodies, kits or dyes were from
Molecular Probes, Invitrogen (Grand Island, NY), unless stated otherwise. Specimens were analyzed with a
Zeiss-LSM780 NLO confocal microscope. All EM samples were analyzed with either an FEI Cm100 or a Zeiss
Supra 55 STEM microscope. ATLAS large scale scan generator (Fibics, Canada) was used for large scale imaging (nanotomy). Data sampling is explained online (www.
nanotomy.org). Data analysis was performed with the
Fibics VEviewer, Zeiss Zen software, Huygens Deconvolution software, Imaris, ImageJ and Adobe Photoshop. Image
assembly was done using Adobe Illustrator and Microsoft
Powerpoint. Abbreviations: PBS = Phosphate Buffered
Saline; TBS = Tris Buffered Saline; BSA = Bovine Serum
Albumin; NGS = Normal Goat Serum, RT = Room Temperature.
Animals and cells
Rats - Analysis of Islets of Langerhans cells from rat
pancreas was performed on embedded tissue as described before (27). Macrostomum lignano was maintained
under standard conditions at 20 C degrees. Drosophila
melanogaster w1118 control flies were raised on standard
cornmeal agar food at 25 C. Salivary glands for polytene

chromosome stainings were dissected from third instar larvae, ovaries were collected and dissected from 34 day old
adult females. PANC-1 (human pancreatic carcinoma) cells
were cultured under standard conditions. Cells were plated
on glass-bottom dishes to analyze mitotic cells.
Immuno electron microscopy
Drosophila ovaries and rat pancreatic tissue were fixed
overnight at 4 C in 2% glutaraldehyde buffered in 0.1M cacodylate and PANC-1 cells were fixed for 1 h. For Macrostomum lignano 10% sucrose was added to the fixative. After
fixation all samples were washed in 0.1M cacodylate buffer
and post-fixed in 1% OsO4 / 1.5% K3 Fe(CN6 ) for 3060
min, dehydrated and embedded in epoxy resin according to
standard protocols. 60 nm sections were collected on nickel
grids, treated with 1% periodic acid for 20 min at RT, rinsed
with distilled water (3 5 min, RT) and blocked in blocking buffer (TBS containing 1% BSA, 0.1% Glycine, 0.1%
cold water fish skin gelatin and 5% normal goat serum).
The grids were incubated overnight with monoclonal mouse
1H6 (0.51 g/ml) in blocking buffer with 1% normal goat
serum) at 4 C, rinsed in TBS (3 5 min, RT), followed by
incubation with secondary antibody, conjugated to 10 nm
gold (British Biocell International; 1:50 in blocking buffer)
for 2 h at RT. Samples were rinsed 3x 5 with distilled water
and contrasted with uranyl acetate and lead citrate. 1H6 was
produced in house and is available from MediMabs (Montreal).
Stylonychia immunofluorescence
For G-quadruplex DNA staining using 1H6 Stylonychia
cells were collected on a 30 m gauze, washed with PBS
and resuspended in 1 ml ice-cold nuclei lysis buffer (0.8%
IGEPAL CA-630, 0.3 M sucrose, 15 mM NaCl, 5mM
MgCl2 , 0.1 mM EGTA, 15 mM TrisHCl pH 7.5, 0.5 mM
DTT, 0.1 mM PMSF, 3.6 ng/ml aprotinin). The suspension
was then layered onto a sucrose cushion (1.6 M sucrose, 15
mM NaCl, 5mM MgCl2 , 0.1 mM EGTA, 15 mM TrisHCl
pH 7.5, 0.5 mM DTT, 0.1 mM PMSF, 3.6 ng/ml aprotinin)
and centrifuged at 4000 rpm using a Heraeus Megafuge
for 45 min at 4 C. The supernatant was carefully removed
before nuclei were fixed in PBS with 1% formaldehyde for
10 min at room temperature. They were then washed with
PBS, and subsequently incubated with glycine stop solution, followed by additional washing with PBS. Nuclei were
then incubated 1 h at RT in blocking buffer (PBS with
0.1% BSA and 0.05% Tween 20). 1H6 mouse monoclonal
antibody binding was subsequently allowed for 1 h at RT
(1 g/ml 1H6 in PBS, 0.1%BSA, 0.05% Tween 20, 1%
DMSO). For secondary detection, after washing the nuclei 2x in PBS, rabbit anti-mouse Alexa-Fluor 433 pAbs
(Molecular Probes) were diluted 1:200 in PBS, 0.1%BSA,
0.05% Tween 20, 1% DMSO and incubated 1 h at RT. For
counterstaining propidium iodide was used upon manufacturers suggestion (Molecular Probes). After several washes
using PBS nuclei were mounted using adhesive slides and
Vectashield mounting medium. Immunofluorescence confocal laser scanning microscopy was performed using the
protocol, antibodies and dyes described in detail previously

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and be single stranded in order for G4 DNA to form. In

principle, this could occur during transcription (18), replication (1921) or DNA repair. In addition, G4 DNA could
form at telomeres either at the 3 single strand G-rich overhang (22) or by molecular crowding of duplex DNA (23).
Recently, it was proposed that the transition of duplex DNA
to quadruplex DNA could serve as a reversible cellular signal promoted by negative supercoiling of DNA (13). Despite accumulating evidence supporting a role for G4 RNA
and DNA in diverse biological processes (24,25), detection
of G4 structures in situ has been problematic in part because
suitable reagents to detect G4 structures have been lacking.
We recently described a mouse monoclonal antibody,
1H6, which strongly binds (Kd = 0.3nM) to synthetic interas well as intramolecular G4 DNA structures (26). The
specificity of 1H6 for various G4 DNA structures was validated in vitro and in vivo and epitopes recognized by 1H6
in cells were found to be sensitive to DNAse treatment but
resistant to RNAse. Staining intensity increased following
incubation with G4 stabilizing ligands and all human tissues tested showed strong nuclear staining as assessed by
light microscopy with notable exception of some cells in the
testis.
In our current study we extended our observations with
the 1H6 antibody to include different species and we performed 1H6 immuno-electron microscopy (EM). Surprisingly, we found that staining is very specific for heterochromatin in all species tested and heterochromatic regions of
Drosophila salivary gland polytene chromosomes. Furthermore, we found that staining is not only very weak in some
cells of human testis but also in cells with germline DNA
from ciliates, flatworms and flies.

154 Nucleic Acids Research, 2016, Vol. 44, No. 1

(28). Images were assembled using ImageJ (Rasband, W.S.,

National Institutes of Health, Bethesda, Maryland, USA;
http://rsb.info.nih.gov/ij/) and Adobe Photoshop CS5 software.
Macrostomum lignano immunohistochemistry

Drosophila melanogaster immunohistochemistry

Drosophila ovaries were collected in cold PBS and fixed
in 4% formaldehyde (from methanol-free 16% Formaldehyde Solution, Thermo Scientific) for 3045 min at RT.
Fixed tissue was washed in PBS-T (PBS containing 0.1%
Triton-X-100) for 1 h at RT. To make the DNA more accessible for the 1H6 antibody and reduce unspecific background, the ovaries were permeabilized and blocked for 30
min in PBS-T containing 5%BSA before incubation with
the primary antibodies. The primary antibodies were mouse
1H6 (1.0 g/ml) and rabbit anti-Vasa (a kind gift from P.
Lasko, McGill, Montreal, Canada) used at a 1:500 dilution, overnight at 4 C. After a wash in PBS-T for 1 h at
RT the samples were incubated in secondary antibodies and
dyes for 2 h at RT. The secondary antibodies, conjugated
to Alexa-Fluor-488 or Alexa-Fluor-594, were used at 1:500
dilutions. F-actin was detected with Rhodamin-Phalloidin
(20U/ml) and DNA by staining with DAPI (0.2 g/ml). After one final wash in PBS-T for 1 h at RT the samples were
mounted in 80% glycerol and analyzed.

RESULTS
G4 structures in the ciliate Stylonychia
DNA in the macronucleus of Stylonychia is organized in
short nanochromosomes ranging in size between about
500bp up to 40kbp with an average size of about 2.7
kb. The telomere of each of these nanochromosomes consists of a 20mer of double stranded 5 C4 A4 C4 A4 and
a 3-single stranded overhang of 16 nucleotides with the
sequence G4 T4 G4 T4 (30). With around 16.000 different
nanochromosomes, each of which has 15.000 copies, a single macronucleus contains an estimated 5 108 telomeres
(31). The first direct evidence that ciliate telomeres adopt an
antiparallel intermolecular G4 structure came from studies
using single chain antibodies directed against the parallel
or antiparallel ciliate telomeric G4 structure (32). Telomeric sequences in Stylonychia appear to be organized in foci
bound to a subnuclear structure (33,34) as was suggested by
earlier electron microscopy (EM) studies (35,36) and most
likely adopt an antiparallel G4 structure (37). Replication
of individual nanochromosomes starts at or close to the
telomeres (38) and in the macronucleus it takes place within
hundreds of synchronously firing replication foci, forming
a migrating morphological distinct region, the replication
band (39). Importantly, the replication band could not be
stained with the phage display antibody in support of the
notion that G4 structures are resolved during DNA replication (32).
1H6 staining of replicating macronuclei with 1H6 revealed a staining pattern similar to that previously observed
(32). Foci-like structures were observed in macronuclei during interphase and S-phase, but neither in replication bands
during S-phase, nor in micronuclei with germline DNA
(Figure 1, arrows and asterisks in bottom panels). Of note,
the DNA in replication bands was clearly stained by the
dye DAPI. Telomeres cluster at the nuclear matrix in the
macronucleus via tightly bound proteins such as TEBP
and TEBP (40). Whereas such proteins could limit accessibility of the antibody to potential binding sites, such limitations are not expected during DNA replication when telom-

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Mature worms were pulsed for 30 min with EdU, relaxed

in 1:1 (v/v) MgCl2 .6H2 O (7.14%) - f/2 and fixed in 4%
paraformaldehyde containing 0,1% Trition X-100 for 30
min. Subsequently, worms were washed with f/2 (5x) and
permeabilized for 1 h with PBS containing 0.25% NP-40 followed by blocking overnight in PBS containing 0.25% NP40, 300mM glycine and 5% (w/v) BSA (blocking medium).
S-phase cells were detected using the Click-It Alexa-Fluor488 Imaging Kit, according to the manufacturers specifications. After washing in PBS containing 0.25% NP-40 (3x)
and blocking medium (2x) the worms were incubated with
mouse monoclonal antibody 1H6 at 2.0 g/ml in blocking
medium followed by rabbit anti-phospho-Histone H3 antibody (Millipore, 1:250) for mitotic cell staining, for 24
h at RT in blocking medium (shaking gradually) and kept
overnight at 4 C. Worms were washed with PBS containing
0.25% NP-40 (3x) and blocking medium (2x) followed by
incubation with secondary antibodies (1:1000), conjugated
to Alexa-Fluor-405 and Alexa-Fluor-546 for 4 h at RT in
blocking medium (while shaking). After washing with PBS
containing 0.25% NP-40 (2x) and PBS (3x), the worms were
counterstained with NucRedTM Dead 647 and mounted in
Prolong Gold anti-fade reagent (Molecular probes).
To calculate 1H6 fluorescence of EdU /H3 , Edu+ /H3
and EdU /H3+ cells in whole mount preparation of labeled
worms we used ImageJ software (v 1.49, NIH). An outline
was drawn around a cell of interest and the mean gray value
and integrated density were measured for each fluorescence
channel. In addition, several background readings outside
the cell area were performed. Corrected total cell fluorescence (CTCF) for each fluorescence channel was calculated
as described (29). Finally, the ratio CTCF (1H6) / CTCF
(DAPI) was determined.

Salivary glands for polytene chromosome stainings were

dissected in PBS from third instar larvae followed by fixation for 5 min with 45% acetic acid - 3.7% formaldehyde. Chromosomes were spread by squashing on slides.
Slides were frozen in liquid nitrogen and the coverslips were
snapped off. Slides were blocked in PBS-1% Triton X-100
(PBST) containing 5% BSA for 30 min at room temperature and incubated overnight with primary antibodies in
PBST-1% BSA at 4 C. The primary antibodies were mouse
1H6 (5 g/ml), rabbit anti-HP1 (1:400, a gift from C.P. Verrijzer), rabbit anti-polII (1:200, a gift from C.P. Verrijzer)
and guinea pig anti-SUUR (1:200, a gift from C.P. Verrijzer). The slides then were washed three times for 15 min
each time in PBST, and incubated with the appropriate fluorescent secondary antibodies in PBST-1% BSA for 1h at
room temperature. The slides then were washed three times
in PBST, air dried and mounted in mounting medium with
4 ,6 -diamidino-2-phenylindole (DAPI) counterstain (Vectashield with DAPI; H-1200; Vector Laboratories).

Nucleic Acids Research, 2016, Vol. 44, No. 1 155

Figure 1. Staining of G4 DNA in the macronucleus of Stylonychia lemnea.

Indirect immunofluorescence of Stylonychia using monoclonal antibody
1H6. Shown are different adult animals with a characteristic staining pattern that excludes the replication band (arrows) as well as the micronucleus
(bottom panels, asterisks). The left panels show 1H6 staining, the second
row corresponding DAPI staining and the third column an overlay of 1H6
and DAPI fluorescence. Scale bars: 10 m.

eres dissociate from the nuclear matrix following phosphorylation of TEBP (41,42).
G4 structures in the regenerative flatworm Macrostomum lignano
Unlike all other cells in human tissues, some cells in human
testis were previously found to be unlabeled or only weakly
labeled by 1H6 (26). Such poorly labeled cells were found at
similar positions to cells expressing high levels of Regulator
of Telomere Length 1 (RTEL1) (43), a helicase implicated in
the processing of G4 DNA (43,44). RTEL1 is essential for
proliferative expansion and differentiation of stem cells in
flatworms (45) and we considered the possibility that resolution or suppression of G4 structures is important for stem
cells with regenerative potential. To test this hypothesis we
studied binding of 1H6 to primitive stem cells in two model
organisms: the flatworm Macrostomum lignano (46) (Figure
2) and Drosophila melanogaster (47) (Figure 3). Pluripotent
stem cells in Macrostomum, called neoblasts, can give rise to
somatic cells as well as cells of the germline (48) and stem
cells of the germline are located at well-defined positions in

1H6 staining of Drosophila melanogaster

We next studied G4 DNA in stem cells of the germline in
ovaries of Drosophila melanogaster. Each ovariole, the basic structural unit of the ovary, consists of a germarium and
a string of maturing egg chambers (Figure 3A,B). At the tip
of each germarium (Figure 3B), two to three germline stem
cells (GSCs) and a number of somatic cells can be identified. GSCs undergo asymmetric cell divisions to give rise to
another stem cell as well as a cystoblast (CB) which, upon
further division, produces 2-cell, 4-cell, 8-cell and 16-cell
germline cysts (50). Mature cysts, comprised of 15 nurse
cells (NC) and a presumptive oocyte (O), are covered by
follicle cells (FCs) (Figure 3B). GSCs and mature nurse
cells showed only weak staining by 1H6 (Figure 3F and G).
In contrast, the oocyte nucleus and most somatic cell nuclei were brightly labeled (Figure 3F, Supplementary Figure S2). Reduced 1H6 staining of GSCs was confirmed by
EM, which also showed less heterochromatin per nuclear
surface area in GSCs compared to other cells located in the
germarium (Figure 3C-E, boxed inserts and corresponding
nanotomy scale images).
Staining of Drosophila polytene chromosomes
To further study the link between heterochromatin and G4
DNA we performed 1H6 staining of polytene chromosomes
from Drosophila third instar stage salivary glands. Such
chromosomes contain heterochromatic bands that are typically underreplicated during endoreplication cycles (54
56). 1H6 showed bright staining of pericentric heterochromatin, ANTP-C and BX-C (57) and a large number of additional bands (Figure 4, middle panels). Antibodies to HP1

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the ovaries of Drosophila (49,50). Immuno-EM of Macrostomum lignano showed that 1H6 antibody binds primarily
to nuclear heterochromatin (Figure 2). This is illustrated for
an epidermal cell and a neoblast (51) in respectively Figure 2C and D. Specific cell types in Macrostomum lignano
were also identified following 1H6 staining using confocal
microscopy. Neoblasts and germline cells, the only proliferating cells in adult animals (52), were identified by incorporation of the nucleoside analog EdU to label S-phase cells
and by staining for phospho-histone H3 to identify mitotic
cells. While most nuclei in adult Macrostomum were brightly
labeled by 1H6, staining of neoblasts and proliferating cells
in the germline was much weaker (Figure 2E-F and Supplementary Figure S1 and Movies 1 and 2). Fluorescence measurements of individual nuclei confirmed a significant difference in 1H6 fluorescence between proliferating and quiescent cells (ratio of integrated 1H6 fluorescence intensity
relative to DAPI fluorescence intensity standard deviation for somatic, EdU /H3 cells = 1.04 0.12; S-phase
EdU+ /H3 cells = 0.65 0.05 and M-phase EdU /H3+
cells = 0.58 0.10; n = 5; P < 0.05). Not all neoblast have
the same developmental potential (53) and some of the differences in 1H6 staining between EdU+ /H3 cells could
reflect neoblast lineage commitment or differentiation. We
conclude that neoblasts and cells of the germline in Macrostomum show less 1H6 staining and that heterochromatin
staining and 1H6 staining appear to be correlated in this
species.

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Figure 2. Weak 1H6 staining of proliferating cells in adult Macrostomum. (A) Bright-field image and schematic overview of Macrostomum lignano. T:
Testis, O: Ovary, E: Egg and S: Stylet. (B) False-colored overview of 1H6 immuno-EM nanotomy data of Figure 2 available at http://www.nanotomy.org/.
Red: Epidermis; Blue: Gut; Yellow: Testis Tip; Green: Testis; Orange: Ovary; Purple: somatic stem cells (neoblasts). (CD) EM images of an epidermal
cell (C, C) and a neoblast (D, D) following labeling with 1H6 antibody. Boxed areas in B are shown in C and D. Boxed area in C and D are shown in C
and D. For clarity, the immunogold particles are marked manually with yellow dots in C and D (raw data available online). (E) Gray scale images of a
section through the germline. stained with 1H6, EdU (S-phase cells), phospho-H3 (mitotic cells) and DNA. (F) Colored overlays of sections shown in E.
Higher magnifications of the boxed area in F are shown in Supplementary Figure S3. Scale bars: A: 100 m, B: 30 m; C, D: 2 m; EF: 20 m.

co-localized in part with 1H6, in support of enrichment

of G4 structures in heterochromatin (Figure 4A and B).
Antibodies specific for the Suppressor of Underreplication
(SUUR) protein showed an even more striking overlap with
the bands labeled by 1H6 although some clear differences
were also noted (Figure 4C and C, Figure 5A and A, arrows). SUUR is a SNF2 domain-containing protein that

marks regions of heterochromatin and late (under-) replication (56,58,59). In contrast, bands labeled with 1H6 appeared mutually exclusive with sites of active transcription
labeled by antibodies specific for RNA polymerase II (Supplementary Figure S3B). As a control we found that binding
of 1H6 to polytene chromosomes could be inhibited by addition of an 10-fold molar excess of synthetic G4 DNA to

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Figure 3. Germline stem cells in Drosophila contain less heterochromatin and stain weaker with 1H6 compared to most other cells. (A) Schematic diagram
of a Drosophila ovariole up to stage 10 of egg chamber development. Green: follicle cells; light blue: nurse cells; yellow: developing oocyte. (B) Schematic
of the germarium. TF: Terminal Filament; CC = Cap Cells; GSC: Germline Stem Cell; CB: Cystoblasts; EC: Escort Cells; GC: Germline Cysts; FSC:
Follicle Stem Cells; FC: Follicle Cells; NC: Nurse Cells; O: Oocyte. (CE) Overview (C,C) and close-up (D,E) images of a germarium, processed for
EM and immunogold-labeled with 1H6 antibody. Nanotomy of Figure 2 shown in C is available online at http://www.nanotomy.org). (C,C) Overview
of the germarium up to stage 3. Individual cell types, identified to the best of our knowledge, were false-colored with Photoshop to match the schematic
shown in B. Close-up snapshots shown in D and E correspond to the red-colored boxes in C and depict a Germline Stem Cell (D) and Germline Cyst
Cell (E) and close-ups of their heterochromatic nuclear regions respectively (D and E). Gold-particles, marking the presence of the 1H6-antibody, were
manually marked with yellow dots (raw data available online) in D and E. Note the co-localization with heterochromatin. (F and G) Part of a Drosophila
ovariole (germarium and stage 45, compare with schematic in A, stained with anti-1H6 (green), anti-Vasa, to indicate the germline cells (red) and the
DNA dye DAPI (blue). Higher magnification images in G correspond to the white box shown in F. In the Drosophila germarium 1H6 antibody shows
strong preference for the Terminal Filament, Cap Cells, Escort Cells (indicated in G), Follicle Cells (white arrow heads in F) and the developing oocyte
(red arrows in F). This can also be seen in later stages of egg development (Supplementary Figure S2). In contrast, the Vasa-positive Germline Stem Cells,
Germline Cysts and future Nurse Cells show much weaker 1H6 staining. Scale bars: C: 10 m; DE: 5 m; F: 50 m and G: 10 m.

158 Nucleic Acids Research, 2016, Vol. 44, No. 1

the antibody during labeling (Supplementary Figure S3A).

To further study the relationship between G4 structures and
SUUR we studied SUUR mutants (Figure 5B and C). In
the absence of SUUR 1H6 staining was maintained (Figure
5B) but overexpression of SUUR resulted in complete loss
of 1H6 staining (Figure 5C).
1H6 immuno-electron microscopy of rat pancreatic cells

DISCUSSION

Figure 4. 1H6 staining of polytene chromosomes. (A) Chromosomes

from salivary glands of third instar Drosophila larvae, stained for 1H6
(green) and the DNA dye DAPI (red). Drosophila salivary glands undergo
through multiple rounds of endoreplication, resulting in polytene chromosomes that can easily be visualized with antibody staining and/or dyes.
1H6 staining reveals multiple specific loci on polytene chromosomes including pericentric heterochromatin (Het.) and developmental loci harboring Antennapedia (ANTP-C) and Bithorax Complex (BX-C). The regions of ANTP-C and BX-C, as well as pericentric heterochromatin are
late replicated and underreplicated in polytene chromosomes (57). The
tips of chromosome arms X, 2L, 2R, 3L and 3R are labeled. Scale bar:
25 m. (B) Pericentric heterochromatin is enriched in DNA quadruplexes.
Polytene chromosomes were stained with antibodies raised against Heterochromatin Protein 1 (HP1, red) and G4 DNA (1H6, green). Pericentric
heterochromatin (Het.) is strongly marked with both antibodies appearing
yellow when merged (right panel). Scale bar: 25 m. (CC) G4 DNA colocalizes with the regions of late replication marked by SUUR. Polytene

Here we report that monoclonal antibody 1H6, specific

for G4 DNA (26), has exquisite specificity for heterochromatic areas in the nucleus, condensed DNA in mitotic chromosomes and heterochromatic bands in polytene chromosomes from Drosophila. Striking differences in 1H6 staining were observed between cells of the soma and cells of
the germline in ciliates, flatworms and flies. In these species
G4 staining ranged from low to undetectable (neoblasts
and germline stem cells in all species tested) to easily detectable and very strong (most somatic cells and oocytes in
Drosophila).
Staining of G4 structures using antibodies was first described over a decade ago (32). Compared to the phage display antibodies used in that study, we observed very similar
but stronger staining by the 1H6 antibody of Stylonychia

chromsomes were stained with SUUR (red) and 1H6 (green). The bands,
which are labeled by both 1H6 and anti-SUUR antibodies are indicated
by arrowheads. Note that, although most of the SUUR binding sites are
also stained by 1H6, there are more additional loci stained only by 1H6.
(C) Split, black and white images for 1H6 (left panel) and SUUR (right
panel). Scale bar: 25 m.

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To further study 1H6 binding to mammalian cells, we performed immuno-electron microscopy (EM) using the surface of ultrathin (60nm) tissue sections of rat pancreatic tissue (Figure 6). To allow the analysis of the immunogold label, used to identify 1H6, in the different cell types within
this tissue, we implemented an automated large-scale electron microscopy (EM) approach known as nanotomy allowing analysis of tissue cross-sections at macromolecular
resolution (27,60).
Strikingly, immunogold particles localize almost exclusively to electron-dense areas of heterochromatin in the nuclei of all cells but some gold particles were also present in
mitochondria and in the cytoplasmic side of the endoplasmic reticulum illustrating the resolution of our approach
(Figure 6, Table 1). Previously, we showed that murine and
human metaphase chromosomes were labeled by 1H6 in a
non-random manner (26). Immuno-EM of a cultured pancreatic cell line confirmed 1H6 staining of mitotic chromosomes (Supplementary Figure S4a and b). Examination
of nucleoli in our 1H6 nanotomy data revealed that these
structures, surrounded by heterochromatin labeled by 1H6,
are themselves devoid of G4 DNA staining (Supplementary
Figure S5A). Note that kinetochores, identified by associated microtubules and their typical ultrastructure appearance, are also devoid of gold label (Supplementary Figure
S5B).

Nucleic Acids Research, 2016, Vol. 44, No. 1 159

lemnea: punctate staining of the macronucleus with the exception of the replication band and no staining of the micronucleus with germline DNA (Figure 1). Loss of G4 DNA
during replication is in agreement with the earlier studies in
ciliates (32), suggesting that G4 structures in the macronucleus occurs are only resolved upon DNA replication (61).
Our results support enrichment of G4 structures in heterochromatin. For our immuno-electron microscopy studies cells and tissues were cross-linked with glutaraldehyde
prior to embedding in plastic, sectioning and antibody incubation. In view of this procedure the induction of G4
structures by 1H6 during antibody incubation seems very
unlikely. Cross-reactivity of 1H6 with other, unknown epitopes is more difficult to exclude. For now we assume that

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Figure 5. Loss of G4 structures upon overexpression of SUUR. (AA) In

polytene chromosomes of wild type third instar Drosophila larvae, nearly
all SUUR binding loci co-localize with G4 DNA (arrowheads) labeled
by anti-SUUR (red) and 1H6 (green) antibodies. (A) Split, black and
white images for 1H6 (left panel) and SUUR (right panel). (B) In polytene
chromsomes of larvae homozygous for SUUR mutant allele G4 DNA loci
are retained. Polytene chromsomes were stained with SUUR (red) and 1H6
(green). Note that SUUR staining is absent. (C) In polytene chromosomes
of larvae overexpressing SUUR under the control of salivary gland specific GAL4 driver (sgs3), 1H6 staining is lost. Polytene chromosomes were
stained with SUUR (red) and 1H6 (green). Note the increased intensity of
SUUR staining. Scale bar: 25 m.

1H6 staining reflects the presence of G4 structures that currently remain rather poorly defined. Most likely at least two
different type of G4 structures are recognized by 1H6: stable G4 structures that are present in heterochromatin and
condensed metaphase chromosomes and presumably much
more transient G4 structures that arise during replication
and perhaps other DNA transactions such as transcription
and recombination.
Whether the more stable G4 structures contribute to heterochromatin formation or are formed following heterochromatin formation is currently not known. Both principles
could apply. Once formed, G4 structures could function to
stabilize heterochromatin and thereby stabilize transcriptional silencing (62). This notion is supported by our observation that 1H6 bands in polytene chromosomes from
Drosophila salivary glands are mutually exclusive with sites
of transcription marked by antibodies against RNA polymerase II (Supplementary Figure S3B).
Outside the nucleus we observed sporadic 1H6 immunogold particles in the cytoplasmic site of the endoplasmatic
reticulum and in mitochondria (Table 1 and Figure 6).
These observations suggest that G4 structures are also
present in some RNA species and in mitochondria. The
availability of a monoclonal antibody that works in postembedding immuno-EM should greatly facilitate additional
studies on the role of G4 structures in various biological
processes. Ideally such further studies should also report
raw data in the nanotomy format presented here rather
than showing selected images.
The partial co-localization of G4 structures with the
SUUR protein is of great interest (Figure 5). Loss of SUUR
is known to restore the differences in DNA copy number in
polytene chromosomes (55,58). The observation that 1H6
staining persist in the absence of SUUR suggest that the
formation of G4 structures does not depend on SUUR and
that G4 structures themselves do not block replication. In
contrast, overexpression of SUUR results in loss of 1H6
staining and increased underreplication. The binding of
SUUR to polytene chromosomes was shown to be dynamic
(63) and SUUR was shown to associate with the replication fork (56). Overexpression of SUUR is also known to
stall replication forks and induce a DNA damage response
as shown by accumulation of H2Av co-localizing with
SUUR (64). To reconcile these various findings we propose
that SUUR could play a role in the resolution of G4 structures and competes with other, limiting, proteins or molecular complexes required to resolve G4 structures at the replication fork (65). A possible role for SUUR in the resolution of heterochromatin is supported by previous studies
showing that over-expression of SUUR results in remarkable swellings in polytene chromosomes that are transcriptionally silent (66).
The precise nature of the epitopes recognized by 1H6 in
cells and chromosomes is currently unclear. Epitopes could
form on folded single strands of DNA or RNA or involve
interactions between multiple molecules (Supplementary
Figure S6). Unfortunately our ongoing attempts to characterize the DNA in chromatin that is enriched for 1H6 binding sites have thus far been unsuccessful (26). Whether this
reflects instability of G4 structures under laboratory conditions or a failure to effectively crosslink or amplify the

160 Nucleic Acids Research, 2016, Vol. 44, No. 1

Table 1. Enumeration of the number and location of immunogold particles observed by immune-electron microscopy in islet of Langerhans cells of rat
pancreas. Results of indirect immunogold labeling with 1H6 and an IgG2b isotype control (MOPC-141; Sigma). Raw data of this experiment is available as
Figure 6 online at: http://www.nanotomy.org/). Abbreviations: Exp = experiment; Mab = monoclonal antibody used; [c] = concentration of antibody used
for staining in microgram/ml; n = number of cells analyzed; n gold/cell = average number of gold particles per cell; Het = number of gold particles present
in heterochromatin; Eu = number of gold particles present in euchromatin; H/E ratio = number of gold particles present in heterochromatin relative to
the number of gold particles present in euchromatin; mito = mitochondria; ER = endoplasmatic reticulum; ER cyto = cytoplasmic side of the ER; ER
lumen = luminal side of the ER; ER C/L ratio = ratio of the number of gold particles present in the cytoplasmic versus the luminal side of the ER; ctr =
isotype control antibody

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Figure 6. 1H6 binds to heterochromatin in different cells of rat pancreas. (A) Overview of pancreatic tissue with alpha cells, beta cells, endothelial cells,
epithelial cells and exocrine cells. The different cell types can be recognized as described (www.nanotomy.nl) (27). A high-resolution nanotomy digital
file of Figure 6, were gold-particles can be detected in a Google-earth like analysis, is available online at http://www.nanotomy.org/). The boxed area in
(A) is shown in (B). The boxed area in (B) is shown in (C) and (D). Annotation of the ultrastructure (C) is illustrated in (D) as follows: heterochromatin
blue; euchromatin purple; cytoplasm yellow; extracellular space in green and an adjacent cell (cytoplasm) in pink. The data of panel (C) were processed to
selectively visualize the gold particles (black). Bars: A: 10 m; B: 1 m; C,D: 0.5 m.

Nucleic Acids Research, 2016, Vol. 44, No. 1 161

intriguing possibility and clarify the emerging role of G4

structures in nuclear organization, gene expression, telomere function and cell differentiation.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
ACKNOWLEDGEMENTS
We thank Piet Borst, Tom Cech, Rudolf Jaenisch, Christopher Pearson and Bas van Steensel for comments on the
manuscript, Ester Falconer, Alexander Henderson, EvertJan Uringa, Sarra Merzouk and Diana Spierings for discussions and Nancy Halsema, Inge Kazemier and Sandra
Henkelman for help with experiments.
Author Contributions: R.H., Y.M.M., S.M., N.A.G.,
R.D.K., A.V.R., J.K., A.H.G.W., K.N., A.V.R. J.P. performed experiments, analyzed data and prepared figures.
Y.M.M., S.M., E.B., J.P., H.L., O.C.M.S. and B.N.G.G
helped with the design of the experiments, performed data
analysis and edited the manuscript. P.M.L designed the
research, analyzed data and wrote the manuscript.
FUNDING
Netherlands Organization for Scientific Research
[NWO175-010-2009-023, ZonMW91111006; STW12718;
ALW 86510012]; Canadian Institute of Health Research
[MOP38075]; National Institute of Health [GMH79042];
Russian Science Foundation [14-14-00221 to A.V.R.].
P.M.L. is a recipient of an Advanced ERC grant. Funding for open access charge: University Medical Center
Groningen, the Netherlands.
Conflict of interest statement. None declared.
REFERENCES
1. Jost,K.L., Bertulat,B. and Cardoso,M.C. (2012) Heterochromatin
and gene positioning: inside, outside, any side? Chromosoma, 121,
555563.
2. Passarge,E. (1979) Emil Heitz and the concept of heterochromatin:
longitudinal chromosome differentiation was recognized fifty years
ago. Am. J. Hum. Genet., 31, 106115.
3. Woodcock,C.L. and Ghosh,R.P. (2010) Chromatin higher-order
structure and dynamics. Cold Spring Harb. Perspect. Biol., 2, a000596.
4. Bickmore,W.A. and van Steensel,B. (2013) Genome architecture:
domain organization of interphase chromosomes. Cell, 152,
12701284.
5. Hubner,M.R., Eckersley-Maslin,M.A. and Spector,D.L. (2013)
Chromatin organization and transcriptional regulation. Curr. Opin.
Genet. Dev., 23, 8995.
6. Fussner,E., Ching,R.W. and Bazett-Jones,D.P. (2011) Living without
30nm chromatin fibers. Trends Biochem. Sci., 36, 16.
7. Nishino,Y., Eltsov,M., Joti,Y., Ito,K., Takata,H., Takahashi,Y.,
Hihara,S., Frangakis,A.S., Imamoto,N., Ishikawa,T. et al. (2012)
Human mitotic chromosomes consist predominantly of irregularly
folded nucleosome fibres without a 30-nm chromatin structure.
EMBO J., 31, 16441653.
8. Hansen,J.C. (2012) Human mitotic chromosome structure: what
happened to the 30-nm fibre? EMBO J., 31, 16211623.
9. Sen,D. and Gilbert,W. (1992) Guanine quartet structures. Methods
Enzymol., 211, 191199.
10. Lane,A.N. (2012) The stability of intramolecular DNA
G-quadruplexes compared with other macromolecules. Biochimie, 94,
277286.

Downloaded from http://nar.oxfordjournals.org/ by guest on March 12, 2016

DNA in these structures in a way that is compatible with

fragmentation and construction of sequencing libraries is
currently not known. However, we previously showed that
1H6 reacts with intra- as well as intermolecular G4 DNA
structures (26) and both types of G4 structures could be
present in vivo (Supplementary Figure S6). The G4 structures recognized by 1H6 could also contain RNA and/or
DNA (6769). Whereas the affinity of 1H6 for G4 DNA is
higher than for G4 RNA ((26) and unpublished observations), the distinction between in situ binding of 1H6 to G4
RNA, G4 DNA or hybrid G4 DNA/RNA structures is not
trivial since the activity of various RNAases on (fixed) G4
structures containing RNA remains to be established. Epitopes recognized by 1H6 could also be present in G4 structures composed of two or four partially double stranded
DNA molecules (Supplementary Figure S6).
Previous studies have shown that DNA capable of G4
DNA is not randomly distributed throughout the genome
(70). In plants, G4 motifs are enriched at genes coupled to
energy status and signaling pathways (71). It has been estimated that there are more than 300 000 sites with this potential in the human genome (1416). If the potential to
form G4 structures is not limited to four runs of guanine repeats and includes hybrid DNA/RNA structures with one
or more G-rich transcripts (Supplementary Figure S6), G4
structures could form at many more locations including, for
example, at telomeres in yeast and at telomeric transposons
in Drosophila melanogaster (72). Another possibility is that
molecular crowding, shown to dissociate duplex telomeric
DNA into G4 DNA (23), could induce the formation of intermolecular G4 DNA structures in cells in the absence of
specific RNA transcripts. The molecular characterization of
the G4 structures recognized by 1H6 as well as the further
mapping of 1H6 binding sites in different cells and chromosomes are important objectives for future studies.
Our results indicate that G4 structures are present in
postmitotic cells as well as in mitotic chromosomes. These
observations suggest that G4 structures can exist independent of DNA replication, transcription or recombination.
In general, the presence of G4 structures in cells could
provide a challenge to genome stability. Given the variable presence of G4 structures in different cells such genomic instability could be cell type specific (compare, e.g.
GSC and oocytes in Figure 3). Instability of G-rich DNA
could occur when G4 structures need to be resolved prior
to transcription, replication, repair or homologous recombination. Failure to unwind G4 structures could result
in contraction and expansions of G-rich repeats. Expansions of G-rich repeats could increase the probability of
G4 formation and favor sequestration of genomic regions
around such repeat expansions into silent heterochromatin.
This mechanism provides an attractive explanation for the
reported link between the length of G-rich repeats and
the suppression of globin gene expression in patients with
ATR-X syndrome (73) and the length of (GGGGCC)n and
(CGG)n repeats and suppression of gene expression in neurodegenerative diseases (74,75). It is tempting to speculate
that differences in the stability of G-rich repeats between
somatic cells and cells of the germline are related to differences in the way G4 structures are formed or are processed
in these different cell types. Future studies will examine this

162 Nucleic Acids Research, 2016, Vol. 44, No. 1

32. Schaffitzel,C., Berger,I., Postberg,J., Hanes,J., Lipps,H.J. and

Pluckthun,A. (2001) In vitro generated antibodies specific for
telomeric guanine-quadruplex DNA react with Stylonychia lemnae
macronuclei. Proc. Natl. Acad. Sci. U.S.A., 98, 85728577.
33. Paeschke,K., Simonsson,T., Postberg,J., Rhodes,D. and Lipps,H.J.
(2005) Telomere end-binding proteins control the formation of
G-quadruplex DNA structures in vivo. Nat. Struct. Mol. Biol., 12,
847854.
34. Jonsson,F., Postberg,J., Schaffitzel,C. and Lipps,H.J. (2002)
Organization of the macronuclear gene-sized pieces of stichotrichous
ciliates into a higher order structure via telomere-matrix interactions.
Chromosome Res., 10, 445453.
35. Lipps,H.J. (1980) In vitro aggregation of the gene-sized DNA
molecules of the ciliate Stylonychia mytilus. Proc. Natl. Acad. Sci.
U.S.A., 77, 41044107.
36. Lipps,H.J., Gruissem,W. and Prescott,D.M. (1982) Higher order
DNA structure in macronuclear chromatin of the hypotrichous ciliate
Oxytricha nova. Proc. Natl. Acad. Sci. U.S.A., 79, 24952499.
37. Sundquist,W.I. and Klug,A. (1989) Telomeric DNA dimerizes by
formation of guanine tetrads between hairpin loops. Nature, 342,
825829.
38. Murti,K.G. and Prescott,D.M. (1983) Replication forms of the
gene-sized DNA molecules of hypotrichous ciliates. Mol. Cell. Biol.,
3, 15621566.
39. Postberg,J., Alexandrova,O., Cremer,T. and Lipps,H.J. (2005)
Exploiting nuclear duality of ciliates to analyse topological
requirements for DNA replication and transcription. J. Cell Sci., 118,
39733983.
40. Gottschling,D.E. and Zakian,V.A. (1986) Telomere proteins: specific
recognition and protection of the natural termini of Oxytricha
macronuclear DNA. Cell, 47, 195205.
41. Paeschke,K., Juranek,S., Rhodes,D. and Lipps,H.J. (2008) Cell
cycle-dependent regulation of telomere tethering in the nucleus.
Chromosome Res., 16, 721728.
42. Paeschke,K., Juranek,S., Simonsson,T., Hempel,A., Rhodes,D. and
Lipps,H.J. (2008) Telomerase recruitment by the telomere end
binding protein-beta facilitates G-quadruplex DNA unfolding in
ciliates. Nat. Struct. Mol. Biol., 15, 598604.
43. Ding,H., Schertzer,M., Wu,X., Gertsenstein,M., Selig,S.,
Kammori,M., Pourvali,R., Poon,S., Vulto,I., Chavez,E. et al. (2004)
Regulation of murine telomere length by Rtel: an essential gene
encoding a helicase-like protein. Cell, 117, 873886.
44. Vannier,J.B., Pavicic-Kaltenbrunner,V., Petalcorin,M.I., Ding,H. and
Boulton,S.J. (2012) RTEL1 dismantles T loops and counteracts
telomeric G4-DNA to maintain telomere integrity. Cell, 149,
795806.
45. Wagner,D.E., Ho,J.J. and Reddien,P.W. (2012) Genetic regulators of a
pluripotent adult stem cell system in planarians identified by RNAi
and clonal analysis. Cell Stem Cell, 10, 299311.
46. Ladurner,P., Scharer,L., Salvenmoser,W. and Rieger,R.M. (2005) A
new model organism among the lower Bilateria and the use of digital
microscopy in taxonomy of meiobenthic Platyhelminthes:
Macrostomum lignano, n. sp (Rhabditophora, Macrostomorpha). J.
Zool. Syst. Evol. Res., 43, 114126.
47. Xi,R. and Xie,T. (2005) Stem cell self-renewal controlled by
chromatin remodeling factors. Science, 310, 14871489.
48. Ladurner,P., Rieger,R. and Baguna,J. (2000) Spatial distribution and
differentiation potential of stem cells in hatchlings and adults in the
marine platyhelminth macrostomum sp.: a bromodeoxyuridine
analysis. Dev. Biol., 226, 231241.
49. Lin,H. (2002) The stem-cell niche theory: lessons from flies. Nat. Rev.
Genet., 3, 931940.
50. Spradling,A., Drummond-Barbosa,D. and Kai,T. (2001) Stem cells
find their niche. Nature, 414, 98104.
51. Rieger,R.M., Legniti,A., Ladurner,P., Reiter,D., Asch,E.,
Salvenmoser,W., Schurmann,W. and Peter,R. (1999) Ultrastructure
of neoblasts in microturbellaria: significance for understanding stem
cells in free-living Platyhelminthes. Invert. Reprod. Dev., 35, 127140.
52. Mouton,S., Willems,M., Braeckman,B.P., Egger,B., Ladurner,P.,
Scharer,L. and Borgonie,G. (2009) The free-living flatworm
Macrostomum lignano: a new model organism for ageing research.
Exp. Gerontol., 44, 243249.

Downloaded from http://nar.oxfordjournals.org/ by guest on March 12, 2016

11. Bucek,P., Jaumot,J., Avino,A., Eritja,R. and Gargallo,R. (2009)

pH-Modulated Watson-Crick duplex-quadruplex equilibria of
guanine-rich and cytosine-rich DNA sequences 140 base pairs
upstream of the c-kit transcription initiation site. Chemistry, 15,
1266312671.
12. Rodriguez Lopez,C.M., Guzman Asenjo,B., Lloyd,A.J. and
Wilkinson,M.J. (2010) Direct detection and quantification of
methylation in nucleic acid sequences using high-resolution melting
analysis. Anal. Chem., 82, 91009108.
13. Li,D., Lv,B., Zhang,H., Lee,J.Y. and Li,T. (2014) Positive
supercoiling affiliated with nucleosome formation repairs non-B
DNA structures. Chem. Commun. (Camb.), 50, 1064110644.
14. Tran,P.L., Mergny,J.L. and Alberti,P. (2011) Stability of telomeric
G-quadruplexes. Nucleic acids Res., 39, 32823294.
15. Huppert,J.L. and Balasubramanian,S. (2005) Prevalence of
quadruplexes in the human genome. Nucleic Acids Res., 33,
29082916.
16. Todd,A.K., Johnston,M. and Neidle,S. (2005) Highly prevalent
putative quadruplex sequence motifs in human DNA. Nucleic Acids
Res., 33, 29012907.
17. Maizels,N. and Gray,L.T. (2013) The G4 genome. PLoS Genet., 9,
e1003468.
18. Duquette,M.L., Handa,P., Vincent,J.A., Taylor,A.F. and Maizels,N.
(2004) Intracellular transcription of G-rich DNAs induces formation
of G-loops, novel structures containing G4 DNA. Genes Dev., 18,
16181629.
19. Cheung,I., Schertzer,M., Rose,A. and Lansdorp,P.M. (2002)
Disruption of dog-1 in Caenorhabditis elegans triggers deletions
upstream of guanine-rich DNA. Nat. Genet., 31, 405409.
20. Kruisselbrink,E., Guryev,V., Brouwer,K., Pontier,D.B., Cuppen,E.
and Tijsterman,M. (2008) Mutagenic capacity of endogenous G4
DNA underlies genome instability in FANCJ-defective C. elegans.
Curr. Biol., 18, 900905.
21. Ribeyre,C., Lopes,J., Boule,J.B., Piazza,A., Guedin,A., Zakian,V.A.,
Mergny,J.L. and Nicolas,A. (2009) The yeast Pif1 helicase prevents
genomic instability caused by G-quadruplex-forming CEB1
sequences in vivo. PLoS Genet., 5, e1000475.
22. Bonetti,D., Martina,M., Falcettoni,M. and Longhese,M.P. (2013)
Telomere-end processing: mechanisms and regulation. Chromosoma,
123, 5766.
23. Miyoshi,D., Matsumura,S., Nakano,S. and Sugimoto,N. (2004)
Duplex dissociation of telomere DNAs induced by molecular
crowding. J. Am. Chem. Soc., 126, 165169.
24. Bochman,M.L., Paeschke,K. and Zakian,V.A. (2012) DNA
secondary structures: stability and function of G-quadruplex
structures. Nat. Rev. Genet., 13, 770780.
25. Wolfe,A.L., Singh,K., Zhong,Y., Drewe,P., Rajasekhar,V.K.,
Sanghvi,V.R., Mavrakis,K.J., Jiang,M., Roderick,J.E., Van der
Meulen,J. et al. (2014) RNA G-quadruplexes cause eIF4A-dependent
oncogene translation in cancer. Nature, 513, 6570.
26. Henderson,A., Wu,Y., Huang,Y.C., Chavez,E.A., Platt,J.,
Johnson,F.B., Brosh,R.M. Jr, Sen,D. and Lansdorp,P.M. (2014)
Detection of G-quadruplex DNA in mammalian cells. Nucleic Acids
Res., 42, 860869.
27. Ravelli,R.B., Kalicharan,R.D., Avramut,M.C., Sjollema,K.A.,
Pronk,J.W., Dijk,F., Koster,A.J., Visser,J.T., Faas,F.G. and
Giepmans,B.N. (2013) Destruction of tissue, cells and organelles in
type 1 diabetic rats presented at macromolecular resolution. Sci. Rep.,
3, 1804.
28. Postberg,J., Heyse,K., Cremer,M., Cremer,T. and Lipps,H.J. (2008)
Spatial and temporal plasticity of chromatin during programmed
DNA-reorganization in Stylonychia macronuclear development.
Epigenetics Chromatin, 1, 3.
29. McCloy,R.A., Rogers,S., Caldon,C.E., Lorca,T., Castro,A. and
Burgess,A. (2014) Partial inhibition of Cdk1 in G 2 phase overrides
the SAC and decouples mitotic events. Cell Cycle, 13, 14001412.
30. Klobutcher,L.A., Swanton,M.T., Donini,P. and Prescott,D.M. (1981)
All gene-sized DNA molecules in four species of hypotrichs have the
same terminal sequence and an unusual 3 terminus. Proc. Natl. Acad.
Sci. U.S.A., 78, 30153019.
31. Aeschlimann,S.H., Jonsson,F., Postberg,J., Stover,N.A., Petera,R.L.,
Lipps,H.J., Nowacki,M. and Swart,E.C. (2014) The draft assembly of
the radically organized Stylonychia lemnae macronuclear genome.
Genome Biol. Evol., 6, 17071723.

Nucleic Acids Research, 2016, Vol. 44, No. 1 163

65. Lopes,J., Piazza,A., Bermejo,R., Kriegsman,B., Colosio,A.,

Teulade-Fichou,M.P., Foiani,M. and Nicolas,A. (2011)
G-quadruplex-induced instability during leading-strand replication.
EMBO J., 30, 40334046.
66. Zhimulev,I.F., Belyaeva,E.S., Semeshin,V.F., Shloma,V.V.,
Makunin,I.V. and Volkova,E.I. (2003) Overexpression of the SuUR
gene induces reversible modifications at pericentric, telomeric and
intercalary heterochromatin of Drosophila melanogaster polytene
chromosomes. J. Cell Sci, 116, 169176.
67. Xu,Y., Kimura,T. and Komiyama,M. (2008) Human telomere RNA
and DNA form an intermolecular G-quadruplex. Nucleic Acids
Symp. Ser., 52, 169170.
68. Wanrooij,P.H., Uhler,J.P., Shi,Y., Westerlund,F., Falkenberg,M. and
Gustafsson,C.M. (2012) A hybrid G-quadruplex structure formed
between RNA and DNA explains the extraordinary stability of the
mitochondrial R-loop. Nucleic Acids Res., 40, 1033410344.
69. Cao,K., Ryvkin,P. and Johnson,F.B. (2012) Computational detection
and analysis of sequences with duplex-derived interstrand
G-quadruplex forming potential. Methods, 57, 310.
70. Eddy,J. and Maizels,N. (2008) Conserved elements with potential to
form polymorphic G-quadruplex structures in the first intron of
human genes. Nucleic Acids Res., 36, 13211333.
71. Andorf,C.M., Kopylov,M., Dobbs,D., Koch,K.E., Stroupe,M.E.,
Lawrence,C.J. and Bass,H.W. (2014) G-quadruplex (G4) motifs in the
maize (Zea mays L.) genome are enriched at specific locations in
thousands of genes coupled to energy status, hypoxia, low sugar, and
nutrient deprivation. Yi Chuan Xue Bao, 41, 627647.
72. Abad,J.P. and Villasante,A. (1999) The 3 non-coding region of the
Drosophila melanogaster HeT-A telomeric retrotransposon contains
sequences with propensity to form G-quadruplex DNA. FEBS Lett.,
453, 5962.
73. Law,M.J., Lower,K.M., Voon,H.P., Hughes,J.R., Garrick,D.,
Viprakasit,V., Mitson,M., De Gobbi,M., Marra,M., Morris,A. et al.
(2010) ATR-X syndrome protein targets tandem repeats and
influences allele-specific expression in a size-dependent manner. Cell,
143, 367378.
74. Haeusler,A.R., Donnelly,C.J., Periz,G., Simko,E.A., Shaw,P.G.,
Kim,M.S., Maragakis,N.J., Troncoso,J.C., Pandey,A., Sattler,R. et al.
(2014) C9orf72 nucleotide repeat structures initiate molecular
cascades of disease. Nature, 507, 195200.
75. Colak,D., Zaninovic,N., Cohen,M.S., Rosenwaks,Z., Yang,W.Y.,
Gerhardt,J., Disney,M.D. and Jaffrey,S.R. (2014) Promoter-bound
trinucleotide repeat mRNA drives epigenetic silencing in fragile X
syndrome. Science, 343, 10021005.

Downloaded from http://nar.oxfordjournals.org/ by guest on March 12, 2016

53. van Wolfswinkel,J.C., Wagner,D.E. and Reddien,P.W. (2014)

Single-cell analysis reveals functionally distinct classes within the
planarian stem cell compartment. Cell Stem Cell, 15, 326339.
54. Rudkin,G.T. (1969) Non replicating DNA in Drosophila. Genetics, 61
(Suppl), 227238.
55. Nordman,J., Li,S., Eng,T., Macalpine,D. and Orr-Weaver,T.L. (2011)
Developmental control of the DNA replication and transcription
programs. Genome Res., 21, 175181.
56. Nordman,J.T., Kozhevnikova,E.N., Verrijzer,C.P., Pindyurin,A.V.,
Andreyeva,E.N., Shloma,V.V., Zhimulev,I.F. and Orr-Weaver,T.L.
(2014) DNA copy-number control through inhibition of replication
fork progression. Cell Rep., 9, 841849.
57. Moshkin,Y.M., Alekseyenko,A.A., Semeshin,V.F., Spierer,A.,
Spierer,P., Makarevich,G.F., Belyaeva,E.S. and Zhimulev,I.F. (2001)
The bithorax complex of Drosophila melanogaster: Underreplication
and morphology in polytene chromosomes. Proc. Natl. Acad. Sci.
U.S.A., 98, 570574.
58. Belyaeva,E.S., Zhimulev,I.F., Volkova,E.I., Alekseyenko,A.A.,
Moshkin,Y.M. and Koryakov,D.E. (1998) Su(UR)ES: a gene
suppressing DNA underreplication in intercalary and pericentric
heterochromatin of Drosophila melanogaster polytene chromosomes.
Proc. Natl. Acad. Sci. U.S.A., 95, 75327537.
59. Pindyurin,A.V., Boldyreva,L.V., Shloma,V.V., Kolesnikova,T.D.,
Pokholkova,G.V., Andreyeva,E.N., Kozhevnikova,E.N.,
Ivanoschuk,I.G., Zarutskaya,E.A., Demakov,S.A. et al. (2008)
Interaction between the Drosophila heterochromatin proteins SUUR
and HP1. J. Cell Sci., 121, 16931703.
60. Faas,F.G., Avramut,M.C., van den Berg,B.M., Mommaas,A.M.,
Koster,A.J. and Ravelli,R.B. (2012) Virtual nanoscopy: generation of
ultra-large high resolution electron microscopy maps. J. Cell Biol.,
198, 457469.
61. Postberg,J., Tsytlonok,M., Sparvoli,D., Rhodes,D. and Lipps,H.J.
(2012) A telomerase-associated RecQ protein-like helicase resolves
telomeric G-quadruplex structures during replication. Gene, 497,
147154.
62. Gray,L.T., Vallur,A.C., Eddy,J. and Maizels,N. (2014) G
quadruplexes are genomewide targets of transcriptional helicases
XPB and XPD. Nat. Chem. Biol., 10, 313318.
63. Kolesnikova,T.D., Posukh,O.V., Andreyeva,E.N., Bebyakina,D.S.,
Ivankin,A.V. and Zhimulev,I.F. (2013) Drosophila SUUR protein
associates with PCNA and binds chromatin in a cell cycle-dependent
manner. Chromosoma, 122, 5566.
64. Andreyeva,E.N., Kolesnikova,T.D., Belyaeva,E.S., Glaser,R.L. and
Zhimulev,I.F. (2008) Local DNA underreplication correlates with
accumulation of phosphorylated H2Av in the Drosophila
melanogaster polytene chromosomes. Chromosome Res., 16, 851862.